GroEL-GroES-mediated protein folding requires an intact central cavity

The chaperonin GroEL is an oligomeric double ring structure that, together with the cochaperonin GroES, assists protein folding. Biochemical analyses indicate that folding occurs in a cis ternary complex in which substrate is sequestered within the GroEL central cavity underneath GroES. Recently, however, studies of GroEL "minichaperones" containing only the apical substrate binding subdomain have questioned the functional importance of substrate encapsulation within GroEL-GroES complexes. Minichaperones were reported to assist folding despite the fact that they are monomeric and therefore cannot form a central cavity. Here we compare directly the folding activity of minichaperones with that of the full GroEL-GroES system. In agreement with earlier studies, minichaperones assist folding of some proteins. However, this effect is observed only under conditions where substantial spontaneous folding is also observed and is indistinguishable from that resulting from addition of the nonchaperone protein alpha-casein. By contrast, the full GroE system efficiently promotes folding of several substrates under conditions where essentially no spontaneous folding is observed. These data argue that the full GroEL folding activity requires the intact GroEL-GroES complex, and in light of previous studies, underscore the importance of substrate encapsulation for providing a folding environment distinct from the bulk solution.     

Dissociation of early folding events from assembly of the human lutropin beta-subunit

The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common alpha-subunit and beta-subunits that confer biological specificity. LHbeta and CGbeta share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHbeta assembles with the alpha-subunit more slowly than does hCGbeta, and only a fraction of the LHbeta synthesized is secreted, whereas CGbeta is secreted efficiently. To understand why the assembly and secretion of these related beta-subunits differ, we studied the folding of LHbeta in CHO cells transfected with either the LHbeta gene alone, or in cells cotransfected with the gene expressing the common alpha-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pbeta1, to the second major folding form, pbeta2, did not differ significantly from the pbeta1-to-pbeta2 conversion of CGbeta, suggesting that variations between the intracellular fates of the two beta-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGbeta, where the folding to pbeta2 results in an assembly-competent product, apparently greater than 90% of the LH pbeta2 recovered from LHbeta-transfected CHO cells was assembly incompetent, accounting for inefficient LHbeta assembly with the alpha-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGbeta, all 12 LHbeta cysteine residues formed S-S linkages as soon as pbeta2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pbeta2 and excess urinary alpha-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGbeta, LHbeta homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective beta-subunits.     

Blood flow lipid transport from viewpoint of protein biochemistry

In accordance with the principles of protein biochemistry, apolipoprotein is the only protein which: 1) forms a protein-lipid complex (PLC) mainly from one class of lipids; 2) determines its functional role; 3) causes dyslipidemia in the genetic destruction of apoproteins or their quantitative ratio. Blood flow lipid transport is based on the functional specificity of their apoproteins; each apoprotein forms a functionally independent PLC; each PLC is formed from one class of lipids; each PLC has one protein-vector; each protein-vector interacts only with receptor. The basis of a united cycle functioning in lipid transport is the difference of primary apoprotein structure. Cholesterol performs an auxiliary function in triglyceride transport by providing circulation in the functional cycle. Lipid transport in blood flow is based, first, on the principles of protein biochemistry and, second, on those of lipidology.     

Measuring individual differences with an information-processing model.

An information-processing model was fitted to individual learning curves for 378 5th–10th graders. Two types of learning materials were examined: paired associates and word definitions. Two parameters of the model displayed reliable individual differences: acquisition rate and long-term retention. Some individual differences also were found in how well students learned definitions under experimenter-paced drill vs student-controlled independent study. Learning scores based on the definitions material had moderately high correlations with traditional aptitude and achievement scores; correlations were lower for paired associates. No significant sex or Spanish-surname vs White group differences were found for the learning scores. Possible classroom applications of the learning scores and parameters are discussed. (16 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Speech perception from an auditory and visual viewpoint.

[Correction Notice: An erratum for this article was reported in Vol 38(1) of Canadian Journal of Psychology Revue Canadienne de Psychologie (see record 2007-03769-001). A programming error occurred in preparing Figure 6. The correct figure looks quite similar to Figure 7, except that the upper eight components are considerably smaller in Figure 6 than in Figure 7. Values quoted in the text remain unchanged.] Processing in the peripheral auditory system of the human ear profoundly alters the characteristics of all acoustic signals impinging on the ear. Some of the 1st-order properties of this peripheral processing are now reasonably well understood: Humans see a heavily overlapped set of filters, with increasingly broader bandwidths at high frequencies, which results in good spectral resolution at low frequencies and good temporal resolution at high frequencies. Results of an examination of speech and music by this system are discussed. An attempt is then made to synthesize several papers on auditory and visual psychophysics, and to speculate on auditory-signal processing analogous to visual-color processing. Several simplified auditory representations of speech are proposed. (French abstract) (25 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The effects of personnel replacement on an information-processing crew.

Which is right: the theory that the human element in a man-machine system is "interchangeable," implying that personnel turnover is irrelevant to the working of the system; or the theory of "crew integrity," implying that performance is hurt by personnel turnover? "The questions posed were specific to information-processing systems and training… . The seven-man information-processing system used in this study was a simple analog of the surveillance and identification sections of the manual aircraft control and warning system (ACW)." Many "crews" were used. They were divided into crews with and without turnover. The indication of performance "was the ratio of the number of task units processed to the number requiring processing." Turnover had bad effects and no training methods tried changed this outcome. From Psyc Abstracts 36:01:3LH91R. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Correcting temperature-sensitive protein folding defects

Recently, we found that different low molecular weight compounds, all known to stabilize proteins in their native conformation, are effective in correcting the temperature-sensitive protein folding defect associated with the deltaF508 cystic fibrosis transmembrane regulator (CFTR) protein. Here we examined whether the folding of other proteins which exhibit temperature-sensitive folding defects also could be corrected via a similar strategy. Cell lines expressing temperature-sensitive mutants of the tumor suppressor protein p53, the viral oncogene protein pp60src, or a ubiquitin activating enzyme E1, were incubated at the nonpermissive temperature (39.5 degrees C) in the presence of glycerol, trimethylamine N-oxide or deuterated water. In each case, the cells exhibited phenotypes similar to those observed when the cells were incubated at the permissive temperature (32.5 degrees C), indicative that the particular protein folding defect had been corrected. These observations, coupled with our earlier work and much older studies in yeast and bacteria, indicate that protein stabilizing agents are effective in vivo for correcting protein folding abnormalities. We suggest that this type of approach may prove to be useful for correcting certain protein folding abnormalities associated with human diseases.     

Easily searched protein folding potentials

In order to calculate the tertiary structure of a protein from its amino acid sequence, the thermodynamic approach requires a potential function of sequence and conformation that has its global minimum at the native conformation for many different proteins. Here we study the behavior of such functions for the simplest model system that still has the essential features of the protein folding problem, namely two-dimensional square lattice chain configurations involving two residue types. First we demonstrate a method for accurately recovering the given contact potential from only a knowledge of which sequences fold to which structures and what the non-native structures are. Second, we show how to derive from the same information more general potential functions having much better positive correlations between potential function value and conformational deviation from the native. These functions consequently permit faster and more reliable searches for the native conformation, given the native sequence. Furthermore, the method for finding such potentials is easily applied to more realistic protein models.     

Toward an information-processing account of individual differences in fraction skills.

The unique associations between 3 families of mathematical knowledge and individual differences in 7th and 8th graders' fraction skills were examined. Procedural knowledge regarding the steps to solve fraction computation problems and conceptual knowledge of part-whole and measurement interpretations of fractions were measured. Math fact knowledge was indexed as the speed of solving single-digit addition and multiplication problems. Conceptual knowledge and procedural knowledge uniquely explained variability in fraction computation solving and fraction word problem set-up accuracy. Only conceptual knowledge uniquely explained individual differences in fraction estimation skills. The findings are discussed within a framework that describes the 3 necessary steps toward an information-processing account of individual differences in fraction skills. Four important limitations to this study were described. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Initial hydrophobic collapse is not necessary for folding RNase A

N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells. The enzyme in this study is the 451 amino acid protein produced by Candida albicans, a yeast responsible for the majority of systemic infections in immuno-compromised humans. NMT activity is essential for vegetative growth, and the structure was determined in order to assist in the discovery of a selective inhibitor of NMT which could be developed as an anti-fungal drug. NMT has no sequence homology with other protein sequences and has a novel alpha/beta fold which shows internal two-fold symmetry, which may be a result of gene duplication. On one face of the protein there is a long, curved, relatively uncharged groove, at the center of which is a deep pocket. The pocket floor is negatively charged due to the vicinity of the C-terminal carboxylate and a nearby conserved glutamic acid residue, which separates the pocket from a cavity. These observations, considered alongside the positions of residues whose mutation affects substrate binding and activity, suggest that the groove and pocket are the sites of substrate binding and the floor of the pocket is the catalytic center.     

Investigation of protein folding by mass spectrometry

Bone repair by regeneration as we know it continues to undergo changes, with advances approaching that may change our treatment of patients with craniofacial deformities and skeletal defects. Perhaps by the turn of the century, patients born with asymmetric deformities due to lack of growth will be treated early in life by skeletal stretching, and then later in life by skeletal distraction that is followed by use of accelerating factors to assist the healing processes. All of these available modalities are part of the regeneration of new bone formation. The future of such changes is very interesting, and our ability to help our patients will be maximized. We may even look back 25 years from now at bone grafting and find it to be obsolete and crude. It is hoped that with the new modalities being developed, we will not deviate from the use of a bone grafting procedure, which is the workhorse of the craniofacial surgeon. Bone grafting is used by all surgeons working on the craniofacial skeleton despite the problems of unpredictability of healing and an inability to calculate what percentage of the original graft will survive. The transplantation issue will be solved. The problems with donor site morbidity will continue. The use of inorganic bone substitutes will continue to have its limitation, particularly in type II wounds, which we as plastic surgeons see in the craniofacial region. As we redefine our approach to skeletal repair, we may look back and find solutions to some of the major problems we have had. The rapid stretch of soft tissue after facial advancement or structural alteration that is accompanied by a relapse due to the elastic recoil of the soft tissue could be eliminated by gradual distraction. The bone will undergo better functional adaptation when it has a gradual change in structure based on adjustment and molding in a gradual fashion. The problem of donor site morbidity and a prediction formula for bone could be resolved with new bone formation in situ by mineralization of the area under repair. Bone healing enhancers are here to stay and their clinical application will produce a far-reaching better final outcome (Fig. 11).     

X-ray solution scattering studies of protein folding

Protein folding is a reaction in which an extended polypeptide chain acquires maximal packing through formation of secondary and tertiary structures. Compactness and shape are, therefore, critical properties characterizing the process of protein folding. Because the stability of the native state is determined by the subtle free energy balance between the native and denatured states, the characterization of the denatured state is also essential to understand the conformational stability of the native state. We show that solution X-ray scattering is the best technique available today to address these problems. Although the structural resolution of the unfolded or compact denatured states elucidated from solution X-ray scattering is low, it provides a variety of information complementary to that obtained by NMR or X-ray crystallography.     

Kinetic evidence for an obligatory intermediate in the folding of the membrane protein bacteriorhodopsin

A photodiode array in conjunction with a rapid stopped-flow mixing method, with a millisecond time resolution, is used here to study the refolding of the membrane protein bacteriorhodopsin from an apoprotein state with a native-like secondary structure in mixed phospholipid/detergent micelles. Refolding to the native state is initiated by the rapid mixing of all-trans-retinal and the apoprotein bacterioopsin in mixed micelles. A lag phase of several seconds is observed in the appearance of the native state, as monitored by the increase in absorbance of the native chromophore. This observation demonstrates unequivocally that an intermediate is obligatory in the formation of bacteriorhodopsin. It is further shown that this intermediate is spectroscopically distinct from free retinal (absorbance maximum approximately 380 nm) and bacteriorhodopsin (absorbance maximum approximately 560 nm) and absorbs maximally at 430 nm. Evidence for the decay of the 430 nm intermediate into bacteriorhodopsin via three distinct parallel pathways is also provided. Taken together, these findings are used to describe a model in which distinct populations of the apoprotein in mixed micelles appear to fold along separate pathways via their corresponding intermediates into the native state. How the results of this study provide new insights into the mechanisms of protein folding is discussed.     

The information-processing theory of mind.

H. A. Simon expresses his thanks for receiving the American Psychological Association citation for outstanding lifetime contribution to psychology. He describes cognitive psychology as a progressive science that, building on behaviorist and Gestalt foundations, has continually broadened and deepened the phenomena that it can predict and explain. (PsycINFO Database Record (c) 2010 APA, all rights reserved)